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. 2011 Aug;7(8):e1002229.
doi: 10.1371/journal.pgen.1002229. Epub 2011 Aug 11.

Glutamine synthetase is a genetic determinant of cell type-specific glutamine independence in breast epithelia

Hsiu-Ni Kung  1 , Jeffrey R Marks, Jen-Tsan Chi
Affiliations

Glutamine synthetase is a genetic determinant of cell type-specific glutamine independence in breast epithelia

Hsiu-Ni Kung et al. PLoS Genet. 2011 Aug.

Abstract

Although significant variations in the metabolic profiles exist among different cells, little is understood in terms of genetic regulations of such cell type-specific metabolic phenotypes and nutrient requirements. While many cancer cells depend on exogenous glutamine for survival to justify the therapeutic targeting of glutamine metabolism, the mechanisms of glutamine dependence and likely response and resistance of such glutamine-targeting strategies among cancers are largely unknown. In this study, we have found a systematic variation in the glutamine dependence among breast tumor subtypes associated with mammary differentiation: basal- but not luminal-type breast cells are more glutamine-dependent and may be susceptible to glutamine-targeting therapeutics. Glutamine independence of luminal-type cells is associated mechanistically with lineage-specific expression of glutamine synthetase (GS). Luminal cells can also rescue basal cells in co-culture without glutamine, indicating a potential for glutamine symbiosis within breast ducts. The luminal-specific expression of GS is directly induced by GATA3 and represses glutaminase expression. Such distinct glutamine dependency and metabolic symbiosis is coupled with the acquisition of the GS and glutamine independence during the mammary differentiation program. Understanding the genetic circuitry governing distinct metabolic patterns is relevant to many symbiotic relationships among different cells and organisms. In addition, the ability of GS to predict patterns of glutamine metabolism and dependency among tumors is also crucial in the rational design and application of glutamine and other metabolic pathway targeted therapies.

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Conflict of interest statement

The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Glutamine addiction phenotypes among different breast cancer cell lines.
(A, B) The normalized cell growth (MTT assay) (A) and viability (trypan blue exclusion assay) (B) of seven indicated breast cancer cell lines (luminal-type: blue, basal-type: green) at different glutamine concentrations. (C) The normalized cell growth of the seven indicated cell lines under control (+G+Q), glutamine deficient (+G-Q), glucose deficient (-G+Q) and glucose/glutamine deficient condition (-G-Q). (D) The percentage of reduction in normalized cellular ATP of the indicated cell lines when cultured in glutamine deficient media for the indicated breast cancer cell lines. (E, F) The glutamine consumption (E) and the intracellular glutamine concentration (F) of the indicated breast cancer cell lines grown in regular media.
Figure 2
Figure 2. Differential expression of genes encoding glutamine-metabolizing enzymes in the basal and luminal breast cancer cell lines.
(A) The heatmap showing the expression levels of probesets for GLUL, GLS and GLS2 in the microarray data of indicated breast cancer cell lines known to be of luminal (blue) and basal (green) type. (B, C, D) The levels of mRNA expression of GLUL (B), GLS (C) and GLS2 (D) of the indicated luminal and breast cell lines determined with real-time PCR. (E) The levels of protein products of GLUL, GLS, and GLS2 in the indicated luminal and basal breast cell lines determined by Western blots.
Figure 3
Figure 3. Persistent differential expression of genes encoding glutamine-metabolizing enzymes in the basal and luminal breast tumors and primary epithelial cells.
(A) The average expression levels were shown for the, GLS and GLS2 in the luminal and basal breast tumors. (B) The heatmap showing the expression levels of probesets for GLUL, GLS and GLS2 in the basal and luminal epithelial cells. (C, D, E) The expression levels determined by real-time PCR were shown for the GLUL (C), GLS (D), and GLS2 (E) in the primary luminal and basal breast epithelial cells. (F) The levels of protein products of GLUL and GLS in the basal and luminal breast epithelial cells by Western blots.
Figure 4
Figure 4. Contribution of luminal expression of GLUL and GATA3 to the glutamine-independence phenotype.
(A) The normalized cell survival of breast cancer cell lines (luminal-type: blue, basal-type: green) with or without treatment with GS inhibitor, L-MS, in the absence of glutamine. (B) The degree of cellular survival under glutamine deprivation for MCF7 (luminal cell) treated with either control or two siRNAs targeting GLUL. (C) The degree of cellular survival under glutamine deprivation for MDAMB231 (basal cell) transfected with either control vector or GLUL overexpression construct. (D) The changes of GLUL, GLS and GLS2 in the mouse mammary epithelia cells transfected with GATA3 from array analysis derived from an independent study . (E) The levels of GLUL in MCF7 cells treated with either control or siRNA targeting GATA3. (F) The levels of GLUL in the MDAMB231 cells transfected with either control vectors or GATA3 expression constructs. (G) The relative survival under glutamine deprivation of MCF7 cells treated with control or two independent siRNAs targeting GATA3. (H) The cell survival rates shown in MDAMB231 cells with overexpression of control vector or GATA3 under glutamine deprivation. (I) The promoter regions of the GLUL with two potential binding sites of GATA3 are shown. (The sequences underlined indicate primer locations.) (J) The enrichment of different promoter regions of GLUL, ER (positive control) and albumin (negative control) which have been immunoprecipitated with GATA3 and control IgG antibodies. (All statistical comparisons: *p<0.05, **p<0.01)
Figure 5
Figure 5. The transcriptional response of breast cancer cell lines to glutamine deprivation.
(A) The heatmap representing the transcriptional response of MCF7 (luminal) and MDAMB231 (basal) cells to glutamine deprivation. (B, C). The predictive probability of the amino acid response (AAR) are shown for the luminal (B, p = 0.4, unpaired t-test) and basal (C, p = 0.0016) cancer cell lines grown under normal (4 mM, Q4) and glutamine-deficient (0 mM, Q0) medium. (D, E) The expression of selected canonical amino acid response genes including XBP1 (D) and DDIT3 (E) in MCF7 and MDAMB231 under indicated concentrations of glutamine with real time RT-PCR.
Figure 6
Figure 6. Potential for glutamine symbiosis between luminal and basal cells.
(A) The protein levels of GS in MCF7 under different concentrations of glutamine. (B, C) The changes of the levels of glutamine in medium (B) and intracellular glutamine concentrations (C) in MCF7 and MDAMB231 cells deprived of glutamine for 12 and 24 h. (D) A diagram illustrating the co-culture systems in E, F. (E, F) The cell viability (E) and the glutamine levels in medium (F) when used to propagate MDAMB231 cells co-cultured with MDAMB231 or MCF7. (G) A diagram illustrating the condition medium model for H, I. (H, I) The cell viability (H) and the glutamine in medium (I) in MDAMB231 cells cultured with fresh medium, MDAMB231 or MCF7 condition medium. (J) A diagram illustrating the model of K. (K) The intracellular glutamine in vector transfected MDAMB231 cells. (L) A diagram illustrating the model of co-culture system for M-O. (M-O) The cell viability (M), the glutamine in medium (N), and intracellular glutamine concentrations (O) in MDAMB231 co-cultured with transfected MDAMB231 cells.
Figure 7
Figure 7. Repression of GLS by GLUL contributes to the polarized expression pattern.
(A, B) The mRNA expression levels of GLS in the indicated MCF7 (luminal, empty) and MDAMB231 (basal, solid) when treated with indicated siRNAs or overexpression constructs. (C, D) The GLUL RNA expression levels in MCF7 (luminal, empty) and MDAMB231 (basal, solid) when treated with siRNA targeted to GLS. (E) GLUL and GLS protein expression levels in MDAMB231 treated with GLUL expression vector. (F, G) The expression of GLS mRNA in MCF7 (luminal, empty) and MDAMB231 (basal, solid) treated with indicated siRNAs or expression construct of GATA3. (H) The GLS protein expression levels in MDAMB231 cells with indicated expression constructs.
Figure 8
Figure 8. A model of glutamine metabolic regulation in different breast cells.
The regulatory mechanisms of glutamine metabolic enzymes based on data from luminal and basal breast cells.
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